Method of reducing egg contamination

ABSTRACT

The present invention relates to Salmonella mutant strains and their use as a vaccine for preventing Salmonella infection, in particular in eggs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/304,484, filed Nov. 26, 2018, which is a is a national stageapplication, filed under 35 U.S.C. §371, of International PatentApplication No. PCT/EP2017/062330, filed on May 23, 2017, which claimspriority to EP 16171540.4, filed May 26, 2016, which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to Salmonella mutant strains and their useas a vaccine for preventing Salmonella infection, in particular in eggs.

SEQUENCE LISTING

A sequence listing in computer readable form (CRF) having file nameSequence_Listing_GHE0028NA.txt (34 kB), created on May 26, 2016, isincorporated by reference into the present disclosure.

BACKGROUND OF THE INVENTION

Salmonellosis is a worldwide occurring disease caused by bacteriabelonging to the genus Salmonella. Salmonella enterica, subspeciesenterica, are Gram-negative bacterial pathogens that are comprised ofmore than 2500 different serovars, of which only a limited number areassociated with poultry. Salmonella enterica serovar Enteritidis (S.Enteritidis; SE) and S. Typhimurium are generally accepted as the mostimportant serovars in chickens, with respect to human public healthsignificance. Chickens infected with the aforementioned serovars appearmainly asymptomatic and continue to shed the bacteria for long periodswith rare cases of systemic disease, except in young chicks. However,these serovars are regularly associated with human infections, whichmostly lead to a self-limiting gastrointestinal disease, and exposure topoultry or poultry products is one of the major risk factors for humaninfection. Majowicz et al. (2010) estimated in 2009 that 93.8 millioncases of gastroenteritis due to Salmonella species occur globally eachyear, with 155,000 deaths. More than 80 million cases were supposed tobe foodborne, and a considerable part of these infections were caused bythe serotype Enteritidis and egg consumption. Infection with S.Enteritidis or S. Typhimurium can become severe, requiring antibiotictreatment or even hospitalization. Hence, a massive burden is stillplaced on both the poultry industry and the healthcare system. Inaddition, with the emergence of multidrug resistant Salmonella strains,antibiotic treatment for human patients is becoming increasinglydifficult. Thus, there is definitely a need for effective measures tocontrol the prevalence of non-host-adapted Salmonella species in poultryflocks.

Eggs are a main vehicle for the pathogen that causes spread to humans.Salmonella can be present on the shell surface due to the presence ofSalmonella in the hen's environment or passage of the egg through thecloaca. In addition, the bacterium can also be contaminating internaleggs after reproductive tract colonization as a consequence of eithershell penetration or colonization of the reproductive tract of layinghens and thus incorporation in the forming egg. In the latter case eggsare a ‘box with Salmonella inside’ that can't be eliminated usinghygienic measures such as egg washing. Several lines of evidence howeversupport the view that egg contamination with SE is more likely to takeplace during the formation of the egg in the reproductive organs than byeggshell penetration. The egg-associated pandemic reached a maximum inthe mid 1990's to the early 2000's. In the European Union, legislationhas been responsible for a serious reduction in Salmonella prevalence atlaying hen farms, eggs and egg products and as a consequence humaninfections due to egg consumption. These legislations forced the memberstates to take action to monitor and control the pathogen, and reductiontargets for prevalence have been produced. Over the past two decades,Salmonella control programs were implemented by the European Union,including that a) that antimicrobials cannot be used to controlSalmonella b) that member states with a prevalence of SalmonellaEnteritidis in commercial laying hens higher than 10% are mandatory tovaccinate and c) that live vaccines can only be used during rearing.Regulation No. 1237/2007 (Anonymous, 2007) laid down restrictions forthe trade of table eggs from flocks infected with Salmonella Enteritidisor Typhimurium. The latter states that eggs from Salmonella Enteritidisor Typhimurium positive flocks must be banned from the market, unlessthey are treated in a manner that guarantees that all Salmonellabacteria are destroyed.

Despite the decline in human cases, salmonellosis still is the secondmost commonly reported zoonotic disease, following campylobacteriosis.Although eggs are no longer the primary food vehicle causingsalmonellosis, it appears that when one considers the risk related tothe different sources weighted according to the tonnage of foodavailable for consumption, the risk of Salmonella infection stillremains the highest when consuming table eggs (EFSA, 2013).

Vaccination of chickens, along with other control measures as part of acomprehensive Salmonella control program, is an important strategy inlowering the prevalence of Salmonella. Vaccination of chickens harnessesthe immune system of the hosts to decrease the levels of Salmonellaspecies that are associated with poultry flocks upon infection ratherthan control disease. The Salmonella vaccines that have been tested aredivided into three categories: live attenuated, inactivated and subunitvaccines (Desin T et al., 2013). Although some commercially availablevaccines are in the killed bacteria form, a few registered S.Enteritidis live vaccines are commercially available for poultry. Theselive vaccines are developed on the principle of either metabolic driftmutations or auxotrophic double-marker mutants obtained through chemicalmutagenesis implicating a higher risk for reverting to virulence (VanImmerseel F et al., 2013). In addition, commercially available vaccinesare developed with the focus on reducing shedding and colonization ofhost tissues such as spleen, liver and caeca, while it is known thatSalmonella colonization in the reproductive tract is generally high andpersistent. In several studies, SE was isolated from the reproductivetissue of infected birds, in the absence of intestinal colonization(Lister, 1988). It has been demonstrated that repeated in vivo passagesthrough the reproductive tissues of chickens increase the ability of anSE strain to induce internal egg contamination, whereas serial passagethrough the liver and the spleen did not affect the ability of thestrain to cause egg contamination (Gast et al., 2003). This is anindication that interaction of SE with the reproductive tissues mayeither induce or select for the expression of microbial propertiesimportant for egg contamination. SE is capable of persistence inreproductive tissues of naturally and experimentally infected hens, eventhough the animals generate an innate and adaptive immune response tothe infection, indicating that the bacteria can reside intracellularlyand escape the host defense mechanisms (Gantois et al., 2009). Thedeposition of Salmonella inside eggs is thus most likely a consequenceof reproductive tissue colonization in infected laying hens (Keller etal., 1995; Methner et al., 1995; Gast & Holt, 2000a). WO2006129090 showsthat vaccination of one-day old chicks with a S. Typhimurium tolC mutantstrain results in a reduced shedding of the S. Typhimurium challengestrain together with a reduced colonization of liver and spleen tissues.However, WO2006129090 is completely silent on colonization of thereproductive tract and egg contamination. Studies documenting protectionagainst egg contamination by vaccination of laying hens are limited(Gantois I et al., 2006). The efficacy of live vaccines in poultry hasbeen tested in experimental and field studies but only a few studieshave demonstrated a partial protective effect of immunization againstegg contamination (Miyamoto T et al., 1999; Woodward MJ et al., 2002;Nassar T J et al., 1994; Hassan J O et al., 1997; Gantois I et al.,2006).

Hence, although some Salmonella vaccines have been shown to be partiallyeffective in reducing the rate of egg contamination, eggs fromvaccinated hens cannot be guaranteed to be Salmonella free. Moreover,vaccine producers only claim a reduction in shedding of the bacteria inthe faeces, not a protection against challenge infection or preventionof egg contamination.

The present invention provides a Salmonella vaccine that specificallycounters the egg contamination and is not merely focused on thereduction of shedding. A further advantage of the present vaccine strainis that it is easy to administer and there is no risk of reversal tovirulence, contrary to some commercial vaccine strains with undefinedmutations.

SUMMARY OF THE INVENTION

It is an objective of the present invention to provide a Salmonellamutant strain, having at least one genetic modification within the tolCgene or within one or more of the resistance-nodulation-division (RND)genes of the efflux pump system. In particular, the Salmonella mutantstrain comprises a genetic modification of the tolC gene or of one orall of the acrAB, acrEF and mdtABC genes. Preferably the geneticmodification is an artificially introduced genetic modification, inparticular resulting in an inactivation of the gene, and more inparticular said modification is a deletion of at least a part of saidgene(s), and more in particular of the complete gene(s).

With the objective to obtain Salmonella mutant strains, the toiC and RNDgene modifications as defined herein, can be applied in wild typeSalmonella serovars. The Salmonella mutant strain as defined and usedherein, includes Salmonella entericaand any serotype of the entericasubspecies, and is typically selected from the group consisting ofSalmonella Enteritidis (S. Enteritidis), S. Typhimurium, S. Hadar, S.Virchow, S. infantis, S. Kentucky, S. Bredeney, S. Agona, S. paratyphi Band S. Gallinarum. In a more particular embodiment said strain isSalmonella ser. Typhimurium, Salmonella ser. Enteriditis, Salmonellaser. Infantis or Salmonella ser. Gallinarum.

It is a further objective of the present invention to provide the use ofa Salmonella mutant strain as described herein, in the manufacture of avaccine and/or for preventing or reducing Salmonella infection in eggs.

In a further embodiment the present invention provides a composition, inparticular a vaccine, comprising the Salmonella strain of the invention,and a pharmaceutically acceptable excipient, carrier and/or diluent, andoptionally an adjuvant.

A further embodiment provides the Salmonella mutant strain, or thecomposition of the present invention for use as a medicament. Moreparticular the invention provides the Salmonella mutant strain e.g. aspart of a vaccine for use in the prevention or inhibition of Salmonellainfection/colonization or a disease caused by such an infection in asubject and/or salmonellosis in humans, and in particular for preventionor (significant) reduction of Salmonella infection in eggs. Anotherembodiment provides the use of the mutant strain or composition of thepresent invention in the treatment or prevention of Salmonellainfection, in particular for immunization of poultry, especially layerhens, against (disease or symptoms caused by) Salmonella infection.

It is also an object of the present invention to provide a method fortreating, preventing, inhibiting and/or reducing the risk of (internal)Salmonella infection in eggs, as well as a method for immunising asubject against Salmonella disease, comprising administering aSalmonella mutant strain or a composition of the present invention, to asubject.

The invention further encompasses a method of producing Salmonella freeeggs by immunising laying hens with the Salmonella mutant providedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The percentage of Salmonella positive samples in spleen (A),caeca (B), oviduct (C) and ovary (D) in non-vaccinated animals andanimals vaccinated at day 1, week 6 and week 16 with SalmonellaEnteritidis 147 ΔtolC or Salmonella Enteritidis 147 ΔacrABacrEFmdtABCstrains, challenged at 3 weeks post-infection with SalmonellaEnteritidis S1400/94, after enrichment. Statistical significantdifferences (p<0.05) in percentage of positive organ samples betweenvaccinated groups and the non-vaccinated control group are marked withan asterix.

FIG. 2: OD values of the ELISA detecting anti-Salmonella LPS antibodiesin the sera of 18 week old laying hens, vaccinated at day 1, week 4 andweek 16 with Salmonella Enteritidis 147 ΔtolC or Salmonella Enteritidis147 ΔacrABacrEFmdtABC. The cut-off OD value is 0.55 and is calculated asthe mean obtained from the sera from the Salmonella free chicks (controlgroup) plus five times the standard deviation.

FIG. 3: Percentage of cloacal swabs positive for the SalmonellaEnteritidis and Salmonella Typhimurium tolC or acrABacrEFmdtABC (Δ7)deletion mutant strains after administration of these strains to one dayold broilers. Broilers were inoculated with both Salmonella Enteritidisand Salmonella Typhimurium tolC deletion mutant strains or withSalmonella Enteritidis and Salmonella Typhimurium acrABacrEFmdtABCdeletion mutant strains on the first day of life. Cloacal swabs werethen weekly taken to monitor shedding of these strains.

FIG. 4: Spleen colonization by Salmonella Enteritidis and SalmonellaTyphimurium tolC or acrABacrEFmdtABC (Δ7) deletion mutant strains afteradministration to one day old broilers. Broilers were inoculated withboth Salmonella Enteritidis and Salmonella Typhimurium tolC deletionmutant strains or with Salmonella Enteritidis and Salmonella TyphimuriumacrABacrEFmdtABC deletion mutant strains on the first day of life.Represented values are log₁₀ CFU/g sample. Samples were taken on day 7,21 and 36. The error bars represent the standard error of the means(SEM).

FIG. 5: Percentage of spleen and caecum samples positive for SalmonellaEnteritidis and Salmonella Typhimurium tolC or acrABacrEFmdtABC (Δ7)deletion mutant strains after enrichment. Broilers were inoculated withboth Salmonella Enteritidis and Salmonella Typhimurium tolC deletionmutant strains or with Salmonella Enteritidis and Salmonella TyphimuriumacrABacrEFmdtABC deletion mutant strains on the first day of life.

FIG. 6: TolC, acrA, acrB, acrD, acrF, acrE, mdsB, mdsA, mdtA, mdtB, andmdtC coding sequences.

FIG. 7: Lohman Brown laying hen body weight after oral inoculation with10⁶ CFU of a Salmonella Gallinarum 9R (SG9R) strain or 10⁶ CFU of aSalmonella Gallinarum tolC (SG tolC) deletion mutant strain on day 35 oflife. Groups treated with either of the strains consisted of 20 animals,and the error bars shown in the figure represent the standard deviationof the mean.

FIG. 8: Necrotic foci scores after post-mortem examination of spleen andliver of Lohmann Brown laying hens that were orally inoculated with aSalmonella Gallinarum 9R (SG9R) strain or a Salmonella Gallinarum ΔtolC(SGtolC) strain. Animals were treated on day 35 of life, liver andspleens were collected and examined on day 63 of life. Necrotic fociscores were determined as described by Matsuda et al. (2011). Necroticfoci scores for the spleen were determined according to the followingmacroscopic findings: score 0: no foci, score 1: fewer than 5 foci,score 2: fewer than 20 foci, score 3: greater than 20 foci. Scores fornecrotic foci in the liver were determined according to macroscopicfindings: score 0: no foci, score 1: fewer than three foci, score 2:fewer than ten foci, score 3: greater than ten foci. Horizontal barsrepresent the mean and the standard error of the mean. No statisticallysignificant differences could be observed between both groups.

FIG. 9: Lohman Brown laying hen spleen and liver weight after oralinoculation with 10⁶ CFU of a Salmonella Gallinarum 9R (SG9R) strain or10⁶ CFU of a Salmonella Gallinarum tolC (SGtolC) deletion mutant strain.Animals were treated on day 35 of life, liver and spleens were collectedand weighed on day 63 of life. Horizontal bars represent the mean andthe standard error of the mean. No statistically significant differencescould be observed between both groups.

FIG. 10: Caecal (A & C) and spleen (B & D) colonization by SalmonellaEnteritidis (A & B) or Salmonella Typhimurium (C & D) wild-type strainson day 7 of age after experimental infection of broiler chickens treatedwith a CI culture. The CI culture was administered on day one of life,and consisted of 10⁸ CFU of a Salmonella Enteritidis ΔacrAbacrEFmdtABCstrain and 10⁸ CFU of a Salmonella Typhimurium ΔacrAbacrEFmdtABC strainadministered simultaneously by oral gavage. The chickens wereexperimentally infected on day 2 of life by administering them 10⁵ CFUof the respective challenge strain by oral gavage. The values shownrepresent log10 of the CFU/g sample. The horizontal lines represent themean, the error bars represent the standard error of mean (SEM). Thenumber of samples equals 10 in all groups.

DESCRIPTION OF THE INVENTION

The present invention relates to a method of preventing Salmonellainfection, in particular Salmonella infection of the reproductive organs(e.g. oviduct, ovary), and even more particular Salmonella infection ineggs. Previous studies demonstrate that the correlation betweenintestinal colonization and colonization of the reproductive tissue isunclear since it has been shown that Salmonella was isolated from thereproductive tissue of infected birds while being absent in theintestinal organs. Hence, existing Salmonella vaccines focusing on areduction in shedding of the bacteria in the faeces will not inevitablyresult in a protection against infection of the reproductive organs, andin particular of (internal) egg contamination.

The invention is based on the finding that vaccines comprisingSalmonella bacteria having a genetic modification in tolC gene or in oneor more of the resistance-nodulation-division (RND) genes of the effluxpump system are able to promote an effective immune response capable ofpreventing or reducing subsequent bacterial infection and/orcolonisation of the reproductive organs, thereby preventing or reducingvertical transmission to and Salmonella contamination ofthe formingeggs. Furthermore, it was demonstrated that said Salmonella bacteria arenot able to infect or colonise the reproductive tract and eggs of thesubjects to whom they are administered, or at least show much reducedability to do so, and hence the bacteria are cleared from the hosthaving provided a suitable and local immunising stimulus. Hence, thedisclosed methods and compositions not only reduce pathogen infection inthe bird but remarkably also reduce incidence of pathogen contaminationin eggs produced by laying birds/hens.

Efflux pumps are found in almost all bacterial species and genesencoding this class of proteins can be located on chromosomes orplasmids. According to their composition, number of transmembranespanning regions, energy sources and substrates, bacterial efflux pumpsare classified into five families: the resistance-nodulation-division(RND) family, the major facilitator superfamily (MFS), the ATP(adenosine triphosphate)-binding cassette (ABC) superfamily, the smallmultidrug resistance (SMR) family (a member of the much largerdrug/metabolite transporter (DMT) superfamily), and the multidrug andtoxic compound extrusion (MATE) family. Except for the RND superfamilywhich is only found in Gram-negative bacteria, efflux systems of theother four families: MFS, ABC, SMR and MATE are widely distributed inboth Gram-positive and negative bacteria. A study by Nishino K. et al.(2006) has shown that S. enterica serovar Typhimurium has ninefunctional drug efflux pumps (AcrAB, AcrD, AcrEF, MdtABC, MdsAB, EmrAB,MdfA, MdtK and MacAB) (see FIG. 1 of Horiyama et al., 2010). Theseefflux pumps in S. enterica are classified into four families on thebasis of sequence similarity: the major facilitator (MF) family (EmrABand MdfA); the RND family (AcrAB, AcrD, AcrEF, MdtABC and MdsAB); themultidrug and toxic compound extrusion (MATE) family (MdtK); and theATP-binding cassette (ABC) family (MacAB). ToIC is a major outermembrane channel involved in siderophore export and is part of themultidrug resistance pumps (MDR).

The present invention provides mutant strains of Salmonella that areuseful as a live or attenuated vaccine for inducing immunologicalprotection against Salmonella, and that are characterized in that theyprevent or reduce Salmonella infection and/or colonization of the hosttissues in a subject, especially of the reproductive organs, and more inparticular in eggs and/or meat. As such, the risk for salmonellosis inhumans is reduced or absent. The mutant strains of the present inventionare characterized in that they contain at least one genetic modificationwithin the tolC gene or within one or more of theresistance-nodulation-division (RND) genes, i.e. acrAB, acrD, acrEF,mdtABC and mdsAB, and especially the acrAB, acrEF and mdtABC genes. Thepresent invention thus provides a Salmonella strain in which at leastone genetic modification within the tolC gene or within one or more ofthe acrAB, acrEF and mdtABC genes was introduced. In particular, thetolC mutant does not comprise any further artificial geneticmodifications within (e.g. deletions of) one or more of the RND genes.In a further embodiment, the acrAB, acrEF and mdtABC mutant comprises anunmodified/complete tolC gene and/or other RND genes. The “geneticmodification” may be an insertion, a deletion, and/or a substitution ofone or more nucleotides in said genes. Such a genetic modificationresults in a (total) decrease in the inherent efflux pump or genefunction of the bacterium. Bacterial efflux pump function may be readilyassayed by means known to those skilled in the art. For example, thelevel of bacterial efflux pump function can be investigated bydetermining the effect of an efflux pump inhibitor on the susceptibilityof a bacterial strain of interest to substrates including antibiotics.Such susceptibility may be analysed by minimum inhibitory concentration(MIC) testing of an antibiotic for test strains in the presence orabsence of efflux pump inhibitor. Preferably a bacterium suitable foruse in accordance with the invention may, for example, have at least 50%less efflux pump function than comparable wild type bacteria,preferably, at least 75% less efflux pump function, more preferably atleast 90% less efflux pump function, and even more preferably 100% lessefflux pump function.

Mutants with inactivated genes or deletion mutants (of the complete geneor (substantial) part thereof) are preferred. The genetic modificationsor mutations may be introduced into the microorganism using any knowntechnique. Preferably, the mutation is a deletion mutation, wheredisruption of the gene is caused by the excision of nucleic acids.Alternatively, mutations may be introduced by the insertion of nucleicacids or by point mutations. Methods for introducing the mutations intothe specific regions will be apparent to the skilled person and arepreferably created using the one step inactivation method described byWanner and Datsenko (2000). Other methods can be applied to achieve asite directed mutagenesis (eg. using suicide plasm ids), however theone-step inactivation method is generally accepted as the best andfastest way to achieve a knock-out deletion mutant.

Preferably, the mutants of the present invention contain a deletion of(at least part of) the tolC gene or one or more of the RND genes of theefflux pump system, including the acrA, acrB, acrD, acrF, acrE, mdsB,mdsA, mdtA, mdtB, or mdtC gene. As used herein, the tolC, acrA, acrB,acrD, acrF, acrE, mdsB, mdsA, mdtA, mdtB, and mdtC gene is meant toinclude any homolog or artificial sequence that is substantiallyidentical, i.e. at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, and preferably 100% identical to the corresponding tolC, acrA,acrB, acrD, acrF, acrE, mdsB, mdsA, mdtA, mdtB, and mdtC gene as foundin Salmonella enterica subsp. enterica serovar Typhimurium str. LT2(NCBI: NC_003197.1 GI:16763390). In said reference sequence the tolCgene is characterized by Gene ID: 1254709 and encodes the ToIC outermembrane channel protein. The acrA gene is characterized by Gene ID:1251996 and encodes the AcrA acridine efflux pump. The acrB gene ischaracterized by Gene ID: 1251995 and encodes the AcrB RND familyacridine efflux pump. The acrD gene is characterized by Gene ID: 1254003and encodes the AcrD RND family aminoglycoside/multidrug efflux pump.The acrF gene is characterized by Gene ID: 1254914 and encodes the AcrFmultidrug efflux protein. The acrE gene is characterized by Gene ID:1254913 and encodes the AcrE multidrug efflux protein. The mdtA gene ischaracterized by Gene ID: 1253647 and encodes the MdtA multidrugresistance protein. The mdtB gene is characterized by Gene ID: 1253648and encodes the MdtB multidrug resistance protein. The mdtC gene ischaracterized by Gene ID: 1253649 and encodes the MdtC multidrugresistance protein. The mdsA gene is characterized by Gene ID: 1251871and encodes the MdsA cation efflux system protein. The mdsB gene ischaracterized by Gene ID: 1251870 and encodes the MdsB cation effluxsystem protein. The nucleic acid sequences of the tolC, acrA, acrB,acrD, acrF, acrE, mdsB, mdsA, mdtA, mdtB, and mdtC genes are given inFIG. 6 (SEQ ID NO: 1-11).

The percentage identity of nucleic acid and polypeptide sequences can becalculated using commercially available algorithms which compare areference sequence with a query sequence. The following programs(provided by the National Center for Biotechnology Information) may beused to determine homologies/identities: BLAST, gapped BLAST, BLASTN andPSI BLAST, which may be used with default parameters.

In one embodiment, the present invention encompasses a Salmonella mutantstrain comprising a deletion of the tolC gene, as compared to thecorresponding wild type sequence as found in Salmonella enterica subsp.enterica serovar Typhimurium str. LT2 (NCBI: NC_003197.1 GI:16763390).In a further embodiment, the present invention encompasses a Salmonellamutant strain comprising a deletion of all of the acrAB, acrEF andmdtABC genes, as compared to the corresponding wild type sequence asfound in Salmonella enterica subsp. enterica serovar Typhimurium str.LT2 (NCBI: NC_003197.1 GI:16763390).

Although any serotype of S. enterica may be used to produce the mutantstrain, in preferred embodiments, the modifications are inserted intoSalmonella serovars most common in poultry, including serovars belongingto serogroup B such as S. Agona, S. Bredeney, S. Paratyphi B, S.Typhimurium, and monophasic strains of S. Typhimurium; serogroup D suchas S. Enteritidis and S. Gallinarum; and serogroup C such as S. Hadar,S. Virchow, S. Infantis, and S. Kentucky. The combination of one or moreof the mutant strains in one composition or vaccine is also envisaged bythe present invention (e.g. mono-, bi-, tri or tetravaccine).

In a particular embodiment said modification(s) are inserted in aSalmonella spp. selected from the group comprising Salmonella Salmonellaenterica subsp. enterica serovar Enteritidis, Salmonella enterica subsp.enterica serovar Typhimurium or Salmonella enterica subsp. entericaserovar Infantis. Salmonella enterica subsp. enterica serovarEnteritidis is a serovar of the D1 serogroup. S. Enteritidis is the mostcommon serovar in the United States and Europe. Salmonella entericasubsp. enterica serovar Typhimurium is a serovar of the B serogroup. S.Typhimurium is a widely distributed serovar, which represent the secondmost common serovar isolated from humans in the United States andEurope. Salmonella enterica subsp. enterica serovar Infantis is aserovar of the C1 serogroup. S. Infantis is commonly found in chickensand broiler flocks.

A “subject” as used herein includes a human or an animal, in particularbirds, more in particular poultry, and even more in particular chickens,especially laying hens (layers), breeders and/or broilers.

“Laying hen” or “egg-laying hen” is a common term for a female chickenthat is kept primarily for laying eggs and includes young chickens thatare reared for egg production. Some chickens are raised for meat (called“broiler” chickens), while others are primarily for eggs (used for humanconsumption). Raising laying hens is a different process than raisingchickens for meat. Broiler chickens typically take less than six weeksto reach slaughter size while most laying hens are kept for one to threelaying cycles (up to 200 weeks) before they are replaced with a newflock. Layers typically start laying eggs around 20 weeks of age. Layerfeeds are formulated for chickens laying table eggs (those used forhuman consumption). Broiler feeds are formulated for those chickensproducing hatching eggs (“breeders”). The diets are basically the same,but the breeder diets typically have slightly more protein and arefortified with extra vitamins for proper embryo development.

In a specific embodiment, the Salmonella mutant strains of the presentinvention are used as attenuated live vaccines. It is well establishedthat live attenuated micro-organisms are highly effective vaccines;immune responses elicited by such vaccines are often of greatermagnitude and of longer duration than those produced by non-replicatingimmunogens. One explanation for this may be that live attenuated strainsestablish limited infections in the host and mimic the early stages ofnatural infection. In addition, unlike killed preparations, livevaccines are often more potent in inducing mucosal immune responses andcell-mediated responses, which may be connected with their ability toreplicate in epithelial cells and antigen-presenting cells, such asmacrophages, respectively. However, concerns remain over the safety ofusing live-attenuated vaccines. There may also be a risk of theattenuated strain reverting to virulence, thus having the potential tocause disease and abortion in the vaccinated animal. However, it wasdemonstrated that the mutant strains of the present invention are safe(no clinical symptoms and not persistently colonizing the host) and donot revert to virulence.

It is an object of the present invention to provide the use of theSalmonella mutant strains of the present invention for preparing amedicament which is employed for the prophylactic and/or therapeutictreatment of Salmonella infection in animals, in particular poultry,more particular chickens, and even more particular in layers. In apreferred embodiment the present invention provides the mutant strainsof Salmonella as defined herein for use as a medicament. In particular,the present invention encompasses the (use of the) mutant strains ofSalmonella as described herein for use in protecting against eggcontamination. Hens' eggs produced by the immunized hens aresubstantially free from Salmonella. Remarkably, the present mutantstrains have been shown to significantly reduce colonization of thereproductive organs. The oviduct can be subdivided into five functionalregions. Starting from the ovary, there are the infundibulum, magnum,isthmus, uterus and vagina. The infundibulum captures the ovulatoryfollicles, the magnum produces the albumen, the isthmus deposits theeggshell membranes, the uterus forms the eggshell and the vagina isinvolved in oviposition. Salmonella colonizing the oviduct could beincorporated into the albumen, the eggshell membranes or the eggshellitself, depending on the site of colonization (magnum, isthmus anduterus, respectively). Although SE has been isolated from both the yolkand the albumen, according to several studies, the albumen is mostfrequently contaminated, pointing to the oviduct tissue as thecolonization site. However, some studies found the yolk to be primarilycontaminated, suggesting the ovary to be the primary colonization site(Gantois et al., 2009). It is thus an aim of the invention to provideSalmonella mutants strains for use in preventing or reducingcolonization/infection of the oviduct tissues and/or the ovary.

In a further embodiment, the Salmonella mutant strains are used tomanufacture a (pharmaceutical) composition, in particular a vaccine,which may be administered to the subject via the parenteral, mucosal ororal route. Live vaccines can be produced using art known procedures andtypically include a (pharmaceutically) acceptable excipient, carrier ordiluent, and optionally an adjuvant.

The present invention provides a pharmaceutical composition or a vaccineagainst Salmonella egg infection comprising:

-   -   one or more of the mutant strains according to the invention;        and    -   a pharmaceutically acceptable carrier or diluent.

The particular pharmaceutically acceptable carriers or diluents employedare not critical to the present invention, and are conventional in theart. Examples of diluents include: buffer for buffering against gastricacid in the stomach, such as citrate buffer (pH 7.0) containing sucrose,bicarbonate buffer (pH 7.0) alone, or bicarbonate buffer (pH 7.0)containing ascorbic acid, lactose, and optionally aspartame. Examples ofcarriers include: proteins, e.g., as found in skimmed milk; sugars, e.g.sucrose; or polyvinylpyrrolidone.

The particular adjuvants employed are not critical to the presentinvention, and are conventional in the art. Examples of adjuvantsinclude, but are not limited to, tensoactive compounds (such as Quil A),mineral salts (such as aluminium hydroxide), micro-organism derivedadjuvants (such as muramyl dipeptide), oil-in-water and water-in-oilemulsions (such as Freund's incomplete adjuvant), particulate antigendelivery systems (such as liposomes, polymeric atmospheres, nanobeads,ISCOMs and ISCOMATRIX), polysaccharides (such as micro-particulateinulin), nucleic acid based adjuvants (such as CpG motivs), cytokines(such as interleukins and interferons), activators of Toll-likereceptors and eurocine L3 en N3 adjuvantia. As is known to the skilledperson, the dose or amount varies according to the route ofadministration. Those skilled in the art may find that the effective(immunizing) dose for a vaccine administered parenterally may be smallerthan a similar vaccine which is administered via drinking water, and thelike. The number of microorganisms that are required to be present inthe formulations can be determined and optimised by the skilled person.However, in general, a subject may be administered approximately10⁴-10¹⁹ colony-forming units (CFUs), preferably between 10⁵-10⁹ CFUs ina single dosage unit, and more preferably between 10⁷-10⁹ CFUs in asingle dosage unit.

As already mentioned hereinbefore, the mutant strains and vaccinecompositions of the present invention may be prepared by knowntechniques.

The choice of particular Salmonella enterica microorganism, can be madeby the skilled person without undue experimentation. A preferredmicroorganism is selected from the group consisting of SalmonellaEnteritidis (S. Enteritidis), S. Typhimurium, S. Hadar, S. Virchow, S.Infantis, S. Kentucky, S. Bredeney, S. Agona, S. paratyphi B and S.Gallinarum. In one embodiment the microorganism is SalmonellaTyphimurium; more in particular the Salmonella Typhimurium strain112910a (Van Parys et al., 2012; De Cort et al., 2014). In a furtherembodiment the microorganism is Salmonella Enteritidis; more particularthe Salmonella Enteritidis strain 147 (Methner et al., 1995; Bohez etal., 2008; De Cort et al., 2013). In an even further embodiment, themicroorganism is either Salmonella Infantis or Salmonella Gallinarum. Ina particular embodiment of the present invention, the mutant strains areeither tolC deletion mutants, or acrABacrEFmdtABC deletion mutants ofSalmonella Typhimurium strain 112910a, or of Salmonella Enteritidisstrain 147.

The Salmonella mutant strains as described herein are especially usefulas vaccines, in particular (for use in a method in order) to prevent or(significantly) reduce Salmonella infection and/or colonization of thehost tissue and/or whereby said mutant strain is capable of preventingor reducing (internal) egg contamination. A further embodiment providesthe composition or vaccine of the present invention for use in theimmunization of chickens, especially layers and broilers, againstSalmonella infection. It is also an object of the present invention toprovide a method for treating, reducing or preventing a Salmonellainfection, comprising administering a Salmonella mutant strain asprovided herein or a composition or vaccine of the present invention, toa subject in need thereof.

Furthermore, the invention is directed to reduce or preventsalmonellosis (e.g. gastroenteritis, vomiting, fever) in humans by theuse of the Salmonella mutant strain and the methods as provided herein.In other words, the invention relates to the use of the Salmonellamutants strain for preventing or reducing egg contamination, e.g. byimmunising or vaccinating hens in order to reduce colonization of thereproductive tissue. By such method, the contamination of the eggs islimited or absent and hence also the risk of salmonellosis and/or thenumber of food borne Salmonella infections in humans. Hence the methodof the invention is especially useful to produce Salmonella free eggs.

In a particular embodiment, the Salmonella Gallinarum mutant strain ischaracterized in that it contains at least one genetic modificationwithin the tolC gene or within one or more of theresistance-nodulation-division (RND) genes, i.e. acrAB, acrD, acrEF,mdtABC and mdsAB, and especially the acrAB, acrEF and mdtABC genes. SaidS. Gallinarum mutant strain is especially useful in protecting layers orbroilers against fowl typhoid, a severe septicaemic disease, inparticular against clinical disease and internal organ colonization byS. Gallinarum. Clinical symptoms include anorexia, diarrhea, anemia, adecreased laying percentage but the major issue is the high mortality itcan induce in both chicks and adult hens

It is recognized that administration of an effective (immunizing) dosemay be achieved by way of a single administration (i.e. administrationof a single dose of a vaccine, said dose constituting an effectivedose), or by way of multiple administration (i.e. administration of twoor more doses of a vaccine, said two or more doses combining toconstitute an effective dose). The use of multiple administrations ofvaccines (for example a primary dose followed by one, two or morebooster doses) is well known, particularly in the context of livevaccines, and is hence an embodiment of the present invention.

Oral administration of the strains or compositions of the invention maybe achieved by inoculation (such as by oral gavage) or by application indrinking water. In one embodiment, the invention relates to (poultry)food comprising the Salmonella mutant(s) as described herein. As analternative to their oral administration, suitably formulated strains orcompositions may be administered to a subject by means of injection. Inparticular, strains or compositions in accordance with the presentinvention may be administered by intramuscular injection, intradermalinjection subcutaneous injection, or intravenous injection. Formulationsfor use in the preparation of injectable vaccines are well known tothose of skill in the art.

Strains or compositions in accordance with the present invention mayalso be administered by inhalation, for example via intranasal spray. Itis well known to provide vaccines by nasal inhalation and suchadministration may be preferred since it lacks many of the undesirableeffects associated with vaccination by injection (such as injection painand the requirement for sterilizing equipment). Suitable nasal sprayformulations which may be used in the preparation of vaccines inaccordance with the present invention will be known to those skilled inthe art.

It has also been shown that effective immunizing dosages of vaccines maybe administered to poultry through the use of whole body sprays. Aerosolimmunization in this manner has been found to be suitable for thegeneration of a systemic immune response, not just a response associatedwith the respiratory tract.

The mutant strains as provided herein can be part of a vaccination kitcomprising a dispensing device and an (immunologically) effective amountof the vaccine strain. The dispensing device is preferably adapted forspray, aerosol delivery or ocular eye drops.

The invention will be described in further details in the followingexamples and embodiments by reference to the enclosed drawings.Particular embodiments and examples are not in any way intended to limitthe scope of the invention as claimed. The rationale of the examplesgiven here for the serotype S. Enteritids are equally well applicable toother Salmonella enterica serotypes infecting poultry, such as forexample S. Typhimurium, S. Hadar, S. Virchow, S. Infantis, S. Kentucky,S. Bredeney, S. Agona, S. Paratyphi B and S. Gallinarum.

EXAMPLES Example 1 Prevention of Egg Contamination by SalmonellaEnteritidis After Oral Vaccination Of Laying Hens with SalmonellaEnteritidis ΔtolC and ΔacrABacrEFmdtABC Mutants

Materials and Methods

Vaccine and Challenge Strains

The vaccine strains ΔtolC and ΔacrABacrEFmdtABC are defined mutants ofSalmonella Enteritidis 147 phage type 4. The wild type strain 147 wasoriginally isolated from egg white and is resistant to streptomycin. Thestrain is known to colonize the gut and internal organs to a high level(Methner, al-Shabibi et al. 1995, Bohez, Dewulf et al. 2008). Allmutations were constructed according to the one step inactivation methodpreviously described by Datsenko and Wanner (Datsenko and Wanner, 2000).

The challenge and vaccine strains were incubated overnight with gentleagitation at 37° C. in Luria Bertani (LB) medium (Sigma, ST. Louis, Mo.,USA). To determine bacterial titers, ten-fold dilutions were plated onbrilliant green agar (BGA, Oxford, Basingstoke, Hampshire, UK) for thechallenge strain. The vaccine strains were plated on LB supplementedwith 1% lactose, 1% phenol red and 100 μg/ml streptomycin to determinethe titer. The vaccine and challenge strains were diluted in HBSS (HanksBalanced Salt Solution, Invitrogen, Paisley, England) to 10⁸ cfu/ml.

Experimental Birds

Ninety (90) day-old Lohmann Brown laying hens (De Biest, Kruishoutem,Belgium) were randomly divided into 3 groups and housed in separateunits. The lighting program provided by the commercial supplier wasimplemented. Commercial feed and drinking water was provided ad libitum.The animal experiment in this study followed the institutionalguidelines for the care and use of laboratory animals and was approvedby the Ethical Committee of the Faculty of Veterinary Medicine, GhentUniversity, Belgium (EC2013/135). Euthanasia was performed with anoverdose of sodium pentobarbital in the wing vein.

Experimental Setup

Two different groups (n=30) were orally immunized at day of birth, at 6weeks of age and at 16 weeks of age through crop instillation of 0.5 mlcontaining 10⁸ cfu of Salmonella Enteritidis 147 ΔtolC (group 1) orSalmonella Enteritidis 147 ΔacrABacrEFmdtABC (group 2). A third group ofbirds (n=30) was kept as non-immunized but Salmonella challengedpositive controls (group 4). At the age of 18 weeks, serum samples weretaken for quantification of anti-Salmonella Enteritidis andanti-Salmonella Typhimurium antibodies in an LPS-ELISA (Desmidt,Ducatelle et al. 1996). At the same time, cloacal swabs were taken ineach group and bacteriologically analyzed for the presence of thevaccine strains. At 21 weeks of age, all the hens were in lay and eggswere collected daily during 3 weeks for bacteriological detection of thevaccine strain in the egg content. At 24 weeks of age, all the animalswere intravenously inoculated in the wing vein with 0.5 ml containing5×10⁷ cfu of the Salmonella Enteritidis strain S1400/94. This protocolwas already used to produce high levels of internal egg contamination(De Buck, Van Immerseel et al. 2004, Gantois, Ducatelle et al. 2006).The eggs were collected daily during 3 weeks and analyzed for thepresence of the challenge strain. Three weeks after challengeinoculation, all the animals were euthanized by an overdose ofpentobarbital in the wing vein. Samples of the spleen, oviduct, ovary,uterus and caecum were aseptically removed for bacteriologicalquantification of challenge and vaccine strain bacteria.

ELISA to Quantify Anti-LPS Antibodies

Serum samples taken at week 18 were analyzed for the levels ofanti-Salmonella LPS antibodies using a previously described indirectELISA protocol (Desmidt et al., 1996). Four 96 well-plates (Sigma, St.Louis, Mo., USA) were coated with 100 μl of an LPS solution (10 μg/ml)in 0.05 M carbonate-bicarbonate (pH 9.6; coating buffer) and incubatedfor 24 hours at 4° C. The LPS was purified from Salmonella EnteritidisPT4, strain 76Sa88 and Salmonella Typhimurium, strain 742Sa91. Theplates were rinsed four times with phosphate buffered saline (PBS,Sigma, St. Louis, Mo., USA) supplemented with 0.1% Tween-20 (Sigma, St.Louis, Mo., USA; washing buffer) between each step. In the first step,100 pl PBS (Sigma, St. Louis, Mo., USA) supplemented with 1% bovineserum albumin (BSA, Sigma, St. Louis, Mo., USA; blocking buffer) wasadded to the wells for one hour at 37° C. The blocking buffer was thenremoved. Secondly, serum samples of animals from the different groupswere diluted in blocking buffer (1:200) and added to the plates (100 μl). As a negative control, serum from a Salmonella free chicken was used.Serum from a chicken that had been infected experimentally withSalmonella Enteritidis PT4, strain 76Sa88, was used as a positivecontrol. The plates were allowed to shake for 2 hours at 37° C. Thirdly,peroxidase-labelled rabbit anti-chicken IgG (100 μl, Sigma, St. Louis,Mo., USA) was diluted (1:2000) in blocking buffer and added to the wellsfor 1 hour and 30 min while shaking at 37° C. Finally 50 μl of TMBsubstrate (Fisher Scientific, Erembodegem, Belgium) was added to thewells. When a blue color started to appear the reaction was blocked with50 μl of sulfuric acid (0.5 M). The absorbance was measured by the ELISAreader at 450 nm. Every sample was analyzed in duplicate The cut-off ODvalue was calculated as the mean obtained from the sera from theSalmonella free chicks (the non-vaccinated birds) plus five times thestandard deviation (OD=0.55).

Bacteriological Examination of the Challenged Birds

The cloacal swabs taken at week 18 were incubated overnight at 37° C. inbuffered peptone water (BPW, Oxoid, Basingstoke, Hampshire, UK).Afterwards a loopful was plated on LB plates supplemented with 1%lactose, 1% phenol red and either 100 μg/ml streptomycin (Sigma,St.Lous, Mo., USA) for the detection of the Salmonella Enteritidis 147Δtol C and ΔacrABacrEFmdtABC vaccine strains. Samples of caecum, spleen,ovary, oviduct and uterus were homogenized in BPW (10% weight/volumesuspensions) and 10-fold dilutions were made in HBSS (Invitrogen,Paisley, England). Six droplets of 20 μl of each dilution were plated onBGA (for quantification of the challenge strain) or on LB supplementedwith 1 lactose, 1% phenol red and the appropriate antibiotics (forquantification of the vaccines). After overnight incubation at 37° C.,the number of cfu/g tissue was determined by counting the number ofbacterial colonies for the appropriate dilution. Samples that testednegative after direct plating for the challenge strain were pre-enrichedin tetrathionate brilliant green broth (Oxoid, Basingstoke, UK) byovernight incubation at 37° C. After incubation, a loopful of thetetrathionate brilliant green broth was plated on BGA.

Egg production and bacteriological examination of eggs Eggs werecollected daily for 6 weeks from week 18 onwards and the egg productionwas determined. Each day, eggs of six chicks per group were pooled inone batch, yielding an egg per batch number that varied between one andsix. Upon collection, lugol solution and 95% ethanol were subsequentlyused to decontaminate the surface of the eggshell. After decontaminationof the eggshell, the eggs were broken aseptically and the total contentof the eggs was pooled and homogenized per batch. A volume of 40 ml ofBPW was added for each egg to the pooled egg content and incubated for48 h at 37° C. To detect the vaccine strains, a loopful of the BPW brothwas plated on LB plates supplemented with 1% lactose, 1% phenol red and100 μg/ml streptomycin. To detect the challenge strain, a loopful of theBPW broth was plated on BGA. Additionally, further enrichment was doneovernight at 37° C. in tetrathionate brilliant green broth and afterincubation, a loopful of broth culture was streaked onto BGA.

Statistical Analysis

GraphPad Prism 5 software was used for statistical analysis. Data of cfuSalmonella/gram tissue of the caecum, spleen, ovary, oviduct and uteruswere log-transformed and analyzed by an anova test followed by a Dunnetpost hoc test to determine differences between the groups. Afterenrichment samples were classified as either positive or negative. AFisher's exact test was used to determine significant differences.Cloacal swabs and batches of eggs were categorized as either positive ornegative. As such a Fisher's exact test was also done to determinesignificant differences. For all tests, differences with p-values below0.05 were considered to be statistically significant.

RESULTS

Detection of Anti-Salmonella LPS Antibodies in Serum

Data derived from the LPS-ELISA show that 26/30 and 19/30 chickscontained anti-Salmonella LPS antibodies in the group of animalsvaccinated with the Salmonella Enteritidis 147 ΔtolC and SalmonellaEnteritidis 147 ΔacrABacrEFmdtABC strain, respectively (FIG. 2).

Analysis of Cloacal Swabs and Eggs for the Presence of Vaccine Strains

No cloacal swabs were found positive in the groups vaccinated with theSalmonella Enteritidis 147 ΔtolC and Salmonella Enteritidis 147ΔacrABacrEFmdtABC strains. No swabs were positive in the non-vaccinatedcontrol group. None of the vaccine strain was isolated from the eggcontent samples.

Clinical Signs and Egg Production After Challenge

Over the whole experiment, there was no reduction in feed and waterintake in either of the groups. The egg production rate after infectionin the unvaccinated control group dropped to 59% in the first weekpost-infection (pi) and raised to 75% and 86% in the second and thirdweek pi. The egg production rate also decreased in the vaccinatedgroups. No significant differences were detected. The egg productionpercentages in the group vaccinated with the ΔtolC strain was 60%, 100%and 90%, and 56%, 70%, 68% for the ΔacrABacrEFmdtABC strain in thefirst, second and third week pi respectively. Some eggs werethin-shelled and malformed during the first week of infection. At theend of the experiment 11 chicks died in the group of animals vaccinatedwith the Salmonella Enteritidis 147 ΔacrABacrEFmdtABC strain because ofcannibalism.

Isolation of the Challenge Strain from Egg Contents

The non-vaccinated hens laid significantly more Salmonella positive eggscompared to the vaccinated animals during the whole 3-week follow-upperiod. Three egg batches were Salmonella positive in the control groupwhile the batches from the vaccine strains were negative after directplating. Not a single positive egg batch was detected for animalsvaccinated with the Salmonella Enteritidis 147 ΔtolC and SalmonellaEnteritidis 147 ΔacrABacrEFmdtABC strains. No positive egg batches werefound in the third week pi.

TABLE 1 The percentage of egg content batches positive for the challengestrain Salmonella Enteritidis S1400/94 in non-vaccinated animals andanimals vaccinated at day 1, week 6 and week 16 with SalmonellaEnteritidis 147 ΔtolC or Salmonella Enteritidis 147 ΔacrABacrEFmdtABCstrains, during the two weeks following infection. Results are shownafter incubation of the egg content in BPW (48 h, 37° C.). Resultsbetween brackets show the percentage of batches positive afterenrichment in tetrathionate brilliant green broth (37° C., overnight).Different superscripts indicate significant differences between thegroups (p < 0.05). Group Week 1 Week 2 Non-vaccinated 70^(a) (74^(a)) 0(17)^(a) ΔtolC 0^(c) (0)^(c) 0 (0)^(c) ΔacrABacrEFmdtABC 0^(c) (0)^(c) 0(0)^(c)

Isolation of the challenge strain from the organs at 3 weekspost-infection No samples were positive at direct plating. Nosignificant differences in Salmonella colonization were seen for theuterus (data not shown). FIG. 1 presents the percentage of Salmonellapositive samples in the spleen, caeca, oviduct and ovary innon-vaccinated animals and animals vaccinated at day 1, week 6 and week16 with either the Salmonella Enteritidis 147 ΔtolC or the SalmonellaEnteritidis 147 ΔacrABacrEFmdtABC strains, at 3 weeks pi with SalmonellaEnteritidis S1400/94 after enrichment. Vaccination with the SalmonellaEnteritidis 147 ΔtolC and ΔacrABacrEFmdtABC strain both significantlydecreased the number of Salmonella positive samples in the spleen,caeca, oviduct and ovary against the control group. Additionally in theΔacrABacrEFmdtABC vaccinated group, the number of Salmonella positivesamples in the oviduct was significantly lower than the group vaccinatedwith ΔtolC.

Example 2 A Salmonella Enteritidis and Salmonella Typhimurium tolC andacrABacrEFmdtABC deletion mutant are safe for use as live vaccinestrains in broilers

Material & Methods

Chickens

One-day-old Ross broiler chickens were obtained from a local hatcheryand housed in isolation. Experimental groups were housed in separaterooms in containers on wood shavings. Commercial feed and drinking waterwere provided ad libitum. Experiments were performed with the permissionof the Ethical Committee of the Faculty of Veterinary Medicine, GhentUniversity, Belgium.

Vaccine Strains

Salmonella Enteritidis 147 StrepR (SE147) is a well-characterized strainoriginally isolated from egg white and was used for the production ofthe deletion mutants (Methner et al. 1995; Methner et al. 1995; Bohez etal. 2008). A spontaneous nalidixic acid-resistant mutant of SalmonellaTyphimurium strain 112910a, originally isolated from a pig stool sample(Van Parys et al. 2012), was used for the production of the otherdeletion mutants. This antibiotic resistance has previously been shownto have no impact on the in vivo results (Barrow et al. 1987). Deletionof the tolC gene or the acrAB, acREF and mdtABC genes was done using theone-step inactivation method described by Datsenko and Wanner (Datsenkoand Wanner 2000; Bohez et al. 2006). This yielded a SalmonellaEnteritidis Strep^(R) tolC deletion mutant, a Salmonella Enteritidis 147Strep^(R) acrAbacrEFmdtABC deletion mutant, a Salmonella TyphimuriumNal^(R) tolC deletion mutant and a Salmonella Typhimurium Nal^(R)acrAbacrEFmdtABC deletion mutant.

Experimental Design

Analysis of the colonisation pattern of Salmonella Enteritidis andSalmonella Typhimurium ΔtolC or ΔacrABacrEFmdtABC mutant strains inbroilers:

evaluation of safety. One hundred and twenty one-day-old chicks weredivided into 2 groups of 60 and each housed in a container of 2,4 m².One group was given 0.5 ml of a mixture containing 2×10⁸ CFU/ml of theSalmonella Enteritidis ΔtolC strain and 2×10⁸ CFU/ml of the SalmonellaTyphimurium ΔtolC strain by oral gavage. The other group was given 0.5ml of a mixture containing 2×10⁸ CFU/ml of the

Salmonella Enteritidis ΔacrAbacrEFmdtABC strain and 2×10⁸ CFU/ml of theSalmonella Typhimurium ΔacrAbacrEFmdtABC strain by oral gavage. Toevaluate colonisation by the deletion mutant strains, their numbers incaecum and spleen were determined for 20 animals at days 7, 21 and 36.Shedding of the strains was evaluated during the experiment bybacteriological analysis of cloacal swabs taken on days 2, 9, 16, 23 and30.

Bacteriological Analysis

Cloacal swabs were directly inoculated on Lysogeny Broth (LB) plateswith 20 μg/ml nalidixic acid (Sigma-Aldrich, St. Louis, Mo., USA) or 100μg/ml streptomycin (Sigma-Aldrich, St. Louis, Mo., USA). Samplesnegative after direct inoculation were pre-enriched in buffered peptonewater (BPW, Oxoid, Basingstoke, England) and incubated overnight at 37°C. One ml of this suspension was further enriched by adding 9 mltetrathionate-brilliant green broth (Merck, Darmstadt, Germany). Afterovernight incubation at 37° C., this suspension was plated on LB platessupplemented with the appropriate antibiotic. Samples of caecum andspleen were homogenized in BPW and 10-fold dilutions were made in HBSS.Six droplets of 20 μl of each dilution were plated on LB platessupplemented with 20 μg/ml nalidixic acid or 100 μg/ml streptomycin.After overnight incubation at 37° C., the number of CFU/g tissue wasdetermined by counting the number of bacterial colonies on the plates.Negative samples were enriched as described above.

Results

Administration of the Salmonella Enteritidis and the SalmonellaTyphimurium tolC deletion mutants and the Salmonella Enteritidis and theSalmonella Typhimurium acrABacrEFmdtABC deletion mutants to one day oldbroilers did not induce clinical symptoms in the animals. In the grouptreated with the Salmonella Enteritidis and the Salmonella TyphimuriumtolC deletion mutants 2 animals died, while in the group treated withthe Salmonella Enteritidis and the Salmonella TyphimuriumacrABacrEFmdtABC deletion mutants 5 animals died. This does not differsignificantly from average mortality (5%) when rearing broilers.(GraphPad Prism 5 software was used for statistical analysis. A Fisher'sexact test (one-sided) was used to analyse mortality rates withindifferently treated groups.)

As shown in FIG. 3, nearly all cloacal swabs taken one day afterinoculation were positive. However, shedding declined quickly with onlya limited number of animals shedding the tolC deletion strains on day16, and no animals were shedding any of the deletion mutant strains fromday 23 onwards.

None of the strains could be detected in the caecum after direct platingon day 7, 21 or 35. In the spleen however, the tolC and theacrABacrEFmdtABC deletion mutant strains colonized the spleen on day 7,and the acrABacrEFmdtABC deletion mutant strains still colonized thespleen on day 21 (FIG. 4). However, by slaughter age (earliest at day36), the Salmonella Enteritidis and Salmonella Typhimurium tolC and theSalmonella Enteritidis and Salmonella Typhimurium acrABacrEFmdtABCdeletion mutant strains could no longer be found in the spleen orcaecum.

Enrichment of caecum and spleen samples confirmed these findings (FIG.5), as both the tolC and acrABacrEFmdtABC deletion mutant strains couldbe found in the spleens of a high percentage of the animals on day 7,and the acrABacrEFmdtABC deletion mutant strains still colonized thespleen on day 21. However, by day 36, none of the strains could still befound in the spleens of any of the animals. In addition, the tolC andacrABacrEFmdtABC deletion mutant strains could only be found in a smallnumber of the caeca after enrichment, and there were no caeca positivefor any of the deletion mutant strains at slaughter age.

These results indicate that both the Salmonella Enteritidis and theSalmonella Typhimurium tolC deletion mutants and the SalmonellaEnteritidis and the Salmonella Typhimurium acrABacrEFmdtABC deletionmutants are safe for use in broilers, and that they are cleared byslaughter age. As a consequence, these strains can thus be used as livevaccine strains in broilers.

Example 3 Evaluation of the Safety of a Salmonella Gallinarum tolCDeletion Mutant Strain for use as a Vaccine Strain Offering ProtectionAgainst Salmonella Gallinarum Infections in Poultry

Material & Methods

Chickens

One-day-old Lohmann Brown laying hens were obtained from a localhatchery and housed in isolation. Experimental groups were housed inseparate rooms in containers of 2.4 m² on wood shavings. Commercial feedand drinking water were provided ad libitum. Experiments were performedwith the permission of the Ethical Committee of the Faculty ofVeterinary Medicine, Ghent University, Belgium.

Vaccine Strains

Salmonella Gallinarum strain 9 (SG9) was used for the production of thedeletion mutants. This strain was originally isolated in the UnitedKingdom (Van Immerseel et al., 2013). Deletion of the tolC gene was doneusing the one-step inactivation method described by Datsenko and Wanner(Datsenko and Wanner, 2000; Bohez et al., 2006). This yielded aSalmonella Gallinarum tolC deletion mutant. In addition, SalmonellaGallinarum 9R (SG9R) was used in this study as well. This strain isfrequently used in practice to control Salmonella Gallinarum infectionsin poultry (Van Immerseel et al., 2013), and was used as a controlstrain to compare the Salmonella Gallinarum tolC deletion mutant strainto.

Experimental Design

Forty one-day old laying hens were randomly divided into two groups oftwenty chickens and housed in separate rooms. They were reared for 5weeks, until the animals were 35 days old. On day 35 of life, allanimals in the first group were orally inoculated with 1 ml of a mixturecontaining 10⁶ CFU/ml of a Salmonella Gallinarum tolC deletion mutant.The other group was orally inoculated with 1 ml of a mixture containing10⁶ CFU/ml of the SG9R strain. The weight of the animals was monitoredfor four weeks, and when the animals were 63 days old (9 weeks) sampleswere taken from liver and spleen to evaluate colonisation by theSalmonella Gallinarum tolC deletion mutant strain and the SG9R strainthrough bacteriological analysis. Post-mortem examination of liver andspleen was performed as well, scoring enlargement and necrotic foci inliver and spleen as described by Matsuda et al. (Matsuda et al., 2011).In addition, the weight of livers and spleens was determined as well.

Bacteriological Analysis

Samples of liver and spleen were homogenized in buffered peptone water(BPW, Oxoid, Basingstoke, England) and 10-fold dilutions were made inHank's Balanced Salt Solution (HBSS, Invitrogen, Paisley, England). Sixdroplets of 20 μl of each dilution were plated on Lysogeny Broth (LB)plates supplemented with 20 μg/ml nalidixic acid or 100 μg/mlstreptomycin. After overnight incubation at 37° C., the number of CFU/gtissue was determined by counting the number of bacterial colonies onthe plates. Negative samples were enriched by adding 9 mltetrathionate-brilliant green broth (Merck, Darmstadt, Germany) to oneml of the samples homogenized in BPW. After overnight incubation at 37°C., this suspension was plated on LB plates supplemented with theappropriate antibiotic.

Statistical Analysis

GraphPad Prism software (Version 5.0, GraphPad Software Inc., La Jolla,Calif.) was used for statistical analysis of the obtained data. AMann-Whitney test was used to analyse the difference in weight and thedifference in enlargement scores and necrotic foci scores between thetwo groups.

Results

Administration of the Salmonella Gallinarum tolC deletion mutant or theSalmonella Gallinarum 9R strain to 5 week old laying hens did not induceclinical symptoms in the animals. No animals died during the experiment,indicating that both strains are severely attenuated when compared towild-type Salmonella Gallinarum strains.

The average body weight of the laying hens before vaccination (on day35) did not differ significantly between the groups (FIG. 7). After oralinoculation with either a SG9R strain or a Salmonella Gallinarum tolCdeletion mutant, no statistical significant differences could beobserved between both groups during the experiment, except on day 51when there was a statistically significant difference between the twogroups. However, this difference was most probably due to the animalsbeing reared in separate rooms, as this was the only day a differencecould be observed and the average weight of the animals in the grouptreated with the Salmonella Gallinarum ΔtolC strain tented to be lowerthroughout the entire experiment, even prior to treatment.

When comparing the necrotic foci scores for the spleen between thedifferently treated groups, no statistically significant differencecould be observed between both groups (FIG. 8). In both groups, only oneliver had more than ten foci. For no other livers necrotic foci could beobserved. As such, there was no statistically significant differencebetween the two groups for liver necrotic foci score (FIG. 8).

No statistically significant differences could be observed whencomparing the average weight of livers and spleens of laying henstreated with a SG9R or a Salmonella Gallinarum ΔtolC strain (FIG. 9). Inaddition, no statistically significant differences could be observedwhen comparing the enlargement scores of liver and spleen. All spleensand livers in the group treated with the SG9R strain received a scoreequal to zero, while one spleen received a score equal to one, and onespleen a score of two in the Salmonella Gallinarum ΔtolC strain treatedgroup. One liver in the group treated with the Salmonella GallinarumΔtolC strain received a score equal to two. However, when comparing thetwo groups, these differences were not statistically significant.

The SG9R and the Salmonella Gallinarum tolC deletion mutant strain couldnot be detected in liver or spleen after bacteriological analysis of thesamples, even after enrichment, indicating that they are cleared fromvaccinated laying hens within 4 weeks after administration if thestrains are administered on day 35 of life.

These results indicate that the Salmonella Gallinarum tolC deletionmutant is an at least as safe vaccine strain as the commonly used SG9Rstrain, as there were no statistically significant differences inremaining virulence between both strains. As a consequence, theSalmonella Gallinarum ΔtolC strain can be used as a live vaccine strainin laying hens.

Example 4 Protection Offered by a Culture Consisting of SalmonellaEnteritidis and Salmonella Typhimurium AacrABacrEFmdtABC Mutant StrainsAgainst Experimental Salmonella Enteritidis and Typhimurium Infection inBroilers: Evaluation of Efficacy

Material & Methods

Chickens

One-day-old Ross 308 broiler chickens were obtained from a localhatchery and housed in isolation. Experimental groups were housed inseparate rooms in containers on wood shavings, while commercial feed anddrinking water were provided ad libitum. The chickens were examineddaily for clinical symptoms following inoculation with Salmonellastrains. Experiments were performed with the permission of the EthicalCommittee of the Faculty of Veterinary Medicine, Ghent University,Belgium.

Salmonella Strains Salmonella Enteritidis 147 strepR (SE147) is awell-characterized strain originally isolated from egg white and wasused for the production of the deletion mutants (Methner et al., 1995a;b; Bohez et al., 2008). A spontaneous nalidixic acid-resistant mutant ofSalmonella Typhimurium strain 112910a, originally isolated from a pigstool sample (Van Parys et al., 2012), was used for the production ofthe Salmonella Typhimurium deletion mutants. This antibiotic resistancehas previously been shown to have no impact on the in vivo results(Barrow et al., 1987). Deletion of the tolC gene or the acrAB, acREF andmdtABC genes was done using the one-step inactivation method describedby Datsenko and Wanner (Datsenko and Wanner, 2000; Bohez et al., 2006).This yielded a Salmonella Enteritidis strepR tolC deletion mutant, aSalmonella Enteritidis 147 StrepR acrABacrEFmdtABC deletion mutant, aSalmonella Typhimurium naIR tolC deletion mutant and a SalmonellaTyphimurium naIR acrABacrEFmdtABC deletion mutant. SalmonellaEnteritidis strain 76Sa88 naIR is a well-characterized nalidixic acidresistant strain which was originally isolated from a poultry farm (VanImmerseel et al., 2002) and was used as a challenge strain in thisstudy. Salmonella Typhimurium MB2136, a streptomycin resistant wild-typestrain originally isolated from swine (De Cort et al., 2015), was alsoused as a challenge strain in this study.

Experimental Design

Forty one-day-old chicks were divided into 4 groups of 10 and eachhoused in a container of 1.2 m2. Two groups were given 0.5 ml of amixture containing 2×108 CFU/ml of the Salmonella EnteritidisΔacrABacrEFmdtABC strain and 2×108 CFU/ml of the Salmonella TyphimuriumΔacrABacrEFmdtABC strain by oral gavage on day 1 of the experiment. Thetwo other groups were given 0.5 ml of Hank's Balanced Salt Solution(HBSS, 14175053, Invitrogen, Paisley, England) by oral gavage as acontrol on day one of the experiment. On day two of the experiment, onecontrol group and one group treated with the CI mixture were given 0.5ml of a solution containing 2×105 CFU/ml of the Salmonella Enteritidis76Sa88 naIR challenge strain by oral gavage, while the other two groupswere challenged by administering 0.5 ml of a solution containing 2×105CFU/ml of the Salmonella Typhimurium MB2136 streptR challenge strain byoral gavage. To evaluate colonization by the challenge strains, theirnumbers in caecum and spleen were determined at day 7 of the experiment.Shedding of the challenge strains was evaluated by bacteriologicalanalysis of cloacal swabs taken on days 3 and 7.

Bacteriological Analysis

Cloacal swabs taken were directly inoculated on xylose lysinedeoxycholate agar (XLD; Oxoid, Basingstoke, England) plates supplementedwith 20 μg/ml nalidixic acid or 100 μg/ml streptomycin. Because theSalmonella Enteritidis ΔacrABacrEFmdtABC strain and the SalmonellaTyphimurium ΔacrABacrEFmdtABC strain are unable to grow on XLD agar, XLDagar was used for the detection of the challenge strains. Samplesnegative after direct inoculation were pre-enriched in buffered peptonewater (BPW; Oxoid, Basingstoke, England) and incubated overnight at 37°C. One ml of this suspension was further enriched by adding 9 mltetrathionate-brilliant green broth (Merck, Darmstadt, Germany). Afterovernight incubation at 37° C., this suspension was plated XLD platessupplemented with the appropriate antibiotic. Samples of caecum andspleen were homogenized in BPW and 10-fold dilutions were made in HBSS.Six droplets of 20 pl of each dilution were plated on XLD plates,supplemented with 20 μg/ml nalidixic acid or 100 μg/ml streptomycin.After overnight incubation at 37° C., the number of CFU/g tissue wasdetermined by counting the number of bacterial colonies on the plates.Negative samples were enriched as described above.

Statistical Analysis

GraphPad Prism software (Version 5.0, GraphPad Software Inc., La Jolla,Calif.) was used for statistical analysis of the obtained data. Achi-square test was used to analyze differences in mortality betweengroups. A Fisher's test was used to analyze statistical differencesbetween groups in the number of Salmonella-positive cloaca swabs and inthe number of spleen and cecum samples positive for Salmonella.Bacterial counts in cecum and spleen were converted into logarithmicform for statistical analysis. Samples of cecum and spleen that werenegative after direct plating were rated as log10=0. Differences betweengroups were analyzed using a Mann-Whitney test. Differences withP-values lower than 0.05 were considered to be significant.

Results

No animals died after during the experiment and as such, there are nostatistical differences in mortality between groups treated with the CIculture and the control groups.

Faecal shedding of the Salmonella Enteritidis challenge strain afterexperimental infection was the same in the control group and the CIculture treated group, with 5 out of 10 chickens shedding the strain inboth groups on day 3 of the experiment. On day 7 of the experiment, only6 out of 10 chickens in the CI treated group were shedding the challengestrain, while 10 out of 10 chickens in the control group were sheddingthe Salmonella Enteritidis challenge strain. Faecal shedding of theSalmonella Typhimurium challenge strain was initially higher in the CItreated group where 5 out of 10 animals were shedding the strain, whilein the control group, only one chicken out of 10 was shedding thestrain. However, on day 7 of the experiment, 10 out of 10 animals wereshedding the Salmonella Typhimurium challenge strain in control group,and 9 out 10 chickens were shedding the challenge strain in the CItreated group.

After direct plating of the caecal samples, the Salmonella Enteritidischallenge strain could not be detected in the group treated with the CIculture (FIG. 10). However, in the control group, the SalmonellaEnteritidis challenge strain could be detected in high numbers inseveral samples. The Salmonella Enteritidis challenge strain could notbe detected in any of the spleen samples, in neither one of the groups.The Salmonella Typhimurium challenge strain could be found insignificantly lower amounts in the group treated with the CI culturewhen compared to the control group (FIG. 10). In the spleen however,there was no significant difference between the treated and theuntreated group in colonization by the Salmonella Typhimurium challengestrain.

After enrichment of the caecal samples, the Salmonella Enteritidischallenge strain could be detected in all samples in both SalmonellaEnteritidis-challenged groups. Similarly, the Salmonella Typhimuriumchallenge strain could be detected in all caecal samples from both thecontrol and the CI treated group (Table 2). After enrichment of thespleen samples, a significantly higher amount of spleens were positivefor the Salmonella Enteritidis challenge strain in the control groupwhen compared to the group treated with the CI culture. There was nosignificant difference in number of spleen samples positive for thechallenge strain between the groups that were experimentally infectedwith the Salmonella Typhimurium challenge strain (Table 2).

TABLE 2 The number of caecal and spleen samples positive for SalmonellaEnteritidis or Salmonella Typhimurium wild-type strains on day 7 of ageafter experimental infection of two days old broiler chickens treatedwith a CI culture. Challenge serotype: Salmonella Enteritidis SalmonellaTyphimurium Group: Control CI treated Control CI treated Caecum10^(a)/10^(b) 10/10 10/10 10/10 Milt 10*/10  2*/10  7/10  8/10^(a)Number of positive samples after enrichment ^(b)Total number ofsamples *Significant difference between control and CI treated groups(P-value < 0.05)

The CI culture was administered on day one of life, and consisted of 108CFU of a Salmonella Enteritidis ΔacrAbacrEFmdtABC strain and 108 CFU ofa Salmonella Typhimurium ΔacrAbacrEFmdtABC strain administeredsimultaneously by oral gavage. The chickens were experimentally infectedon day 2 of life by administering them 105 CFU of the respectivechallenge strain by oral gavage.

Conclusion

A CI culture consisting of the ΔacrABacrEFmdtABC strains is able tooffer protection against Salmonella Enteritidis and Typhimurium afterexperimental infection. As such, these strains can be used to helpreduce Salmonella prevalence in broilers and eventually reduce thenumber of food borne Salmonella infections in humans.

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1. A method of immunizing a subject against Salmonella infectioncomprising administering a Salmonella mutant strain of the serovarSalmonella enterica subsp. Enterica, said mutant strain comprising agenetic modification in the tolC gene or in at least the acrAB, acrEFand mdtABC gene, and wherein said mutant strain is capable of preventingor reducing Salmonella colonization of the reproductive organs in saidhens.
 2. The method according to claim 1, wherein the geneticmodification is a deletion of at least a portion of the tolC gene or theacrAB, acrEF and mdtABC genes, or wherein the genetic modificationresults in the inactivation of said gene(s).
 3. The method according toclaim 1, wherein the Salmonella mutant strain is selected from theserogroup B, C or D.
 4. The method according to claim 1, wherein thestrain is selected from the group consisting of Salmonella entericasubsp. enterica serovar Enteritidis, Salmonella enterica subsp. entericaserovar Typhimurium, Salmonella enterica subsp. enterica serovar Hadar,Salmonella enterica subsp. enterica serovar Virchow, Salmonella entericasubsp. enterica serovar Infantis, Salmonella enterica subsp. entericaserovar Kentucky, Salmonella enterica subsp. enterica serovar Bredeney,Salmonella enterica subsp. enterica serovar Agona, Salmonella entericasubsp. enterica serovar Paratyphi B and Salmonella enterica subsp.enterica serovar Gallinarum.
 5. The method according to claim 1, whereinthe mutant strain is part of a vaccine composition further comprising apharmaceutically acceptable carrier and/or diluent, and optionally anadjuvant.
 6. The method according to claim 1, wherein the mutant strainis part of a formulation suitable for administration by injection,inhalation, or oral administration.
 7. The method according to claim 1,wherein the strain or composition is administered in a prime-boostregimen.
 8. The method according to claim 1, wherein the subject is abroiler hen or a laying hen.
 9. The method according to claim 8, whereinsaid immunized hen produces eggs substantially free of Salmonella.
 10. Amethod of preventing or reducing Salmonella contamination of eggs, saidmethod comprising administering a Salmonella mutant strain comprising agenetic modification in the tolC gene or in at least the acrAB, acrEFand mdtABC gene to layings hens.